Leibniz Institute for Molecular Pharmacology, National Institute of Allergy and Infectious Diseases, University of Newcastle, University of Sydney, US Army Medical Research Institute of Infectious Diseases, Wisconsin National Primate Research Center

Abstract: Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques, but is thought to be nonpathogenic for humans. To date, the SHFV lifecycle is almost completely uncharacterized on the molecular level. Here we describe the first steps of the SHFV lifecycle. Our experiments indicate that SHFV enters target cells by low pH-dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-β-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin, and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knock-out study and electron-microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosis-like pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, α-chymotrypsin, trypsin) abrogated entry, indicating that the SHFV cell-surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFV-related porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component.Simian hemorrhagic fever virus (SHFV) causes highly lethal disease in Asian macaques resembling human illness caused by Ebola or Lassa viruses. However, little is known about SHFV's ecology, molecular biology, and the mechanism by which it causes disease. Results of this study shed light on how SHFV enters its target cells. Using electron microscopy and inhibitors for various cellular pathways, we demonstrate that SHFV invades cells by low pH-dependent, actin-independent endocytosis, with the likely help of a cellular surface protein.